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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1980 Feb;77(2):965–969. doi: 10.1073/pnas.77.2.965

Synthesis and insertion of cytochrome P-450 into endoplasmic reticulum membranes.

S Bar-Nun, G Kreibich, M Adesnik, L Alterman, M Negishi, D D Sabatini
PMCID: PMC348404  PMID: 6767247

Abstract

Treatment of rats with phenobarbital leads to a substantial increase in levels of translatable liver cytochrome P-450 mRNA. This mRNA is primarily associated with ribosomes bound to endoplasmic reticulum membranes which in an in vitro system synthesized approximately 10 times more cytochrome P-450 than did free polysomes from the same animals. Cytochrome P-450 synthesized by rough microsomes in vitro appears to be directly inserted into the membranes because it was not released by a treatment with low detergent concentrations that released albumin and other microsomal content proteins. The amino-terminal amino acid sequence of cytochrome P-450 synthesized in an mRNA-dependent system resembles in hydrophobicity the signal segment of presecretory proteins and therefore may serve to insert the polypeptide into the membrane during synthesis. In contrast to the situation with secretory proteins and several other membrane proteins, however, the putative insertion signal of cytochrome P-450 is not removed by a membrane-associated peptidase and remains in the mature polypeptide.

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Selected References

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  1. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bhat K. S., Padmanaban G. Cytochrome P-450 synthesis in vivo and in a cell-free system from rat liver. FEBS Lett. 1978 May 15;89(2):337–340. doi: 10.1016/0014-5793(78)80250-6. [DOI] [PubMed] [Google Scholar]
  3. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  5. Chyn T. L., Martonosi A. N., Morimoto T., Sabatini D. D. In vitro synthesis of the Ca2+ transport ATPase by ribosomes bound to sarcoplasmic reticulum membranes. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1241–1245. doi: 10.1073/pnas.76.3.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Craft J. A., Cooper M. B., Estall M. R., Rabin B. R. The biosynthesis of cytochrome P450 by rough endoplasmic reticulum in vitro. A significant proportion of newly-biosynthesised cytochrome P450 is resistant to proteolytic digestion in intact vesicles. FEBS Lett. 1979 Feb 15;98(2):403–407. doi: 10.1016/0014-5793(79)80227-6. [DOI] [PubMed] [Google Scholar]
  7. Craft J. A., Cooper M. B., Estall M. R., Rees D. E., Rabin B. R. The role of components of the endoplasmic reticulum in the biosynthesis of cytochrome P-450. Eur J Biochem. 1979 May 15;96(2):379–391. doi: 10.1111/j.1432-1033.1979.tb13050.x. [DOI] [PubMed] [Google Scholar]
  8. Craft J. A., Cooper M. B., Rabin B. R. The biosynthesis of cytochrome P-450 in vitro. FEBS Lett. 1978 Apr 1;88(1):62–66. doi: 10.1016/0014-5793(78)80607-3. [DOI] [PubMed] [Google Scholar]
  9. Emr S. D., Schwartz M., Silhavy T. J. Mutations altering the cellular localization of the phage lambda receptor, an Escherichia coli outer membrane protein. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5802–5806. doi: 10.1073/pnas.75.12.5802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fujii-Kuriyama Y., Negishi M., Mikawa R., Tashiro Y. Biosynthesis of cytochrome P-450 on membrane-bound ribosomes and its subsequent incorporation into rough and smooth microsomes in rat hepatocytes. J Cell Biol. 1979 Jun;81(3):510–519. doi: 10.1083/jcb.81.3.510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Garoff H., Simons K., Dobberstein B. Assembly of the Semliki Forest virus membrane glycoproteins in the membrane of the endoplasmic reticulum in vitro. J Mol Biol. 1978 Oct 5;124(4):587–600. doi: 10.1016/0022-2836(78)90173-0. [DOI] [PubMed] [Google Scholar]
  12. Greenway D. C., MacLennan D. H. Assembly of the sarcoplasmic reticulum. Synthesis of calsequestrin and the Ca2+ + Mg2+ -adenosine triphosphatase on membrane-bound polyribosomes. Can J Biochem. 1978 Jun;56(6):452–456. doi: 10.1139/o78-070. [DOI] [PubMed] [Google Scholar]
  13. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  14. Habener J. F., Rosenblatt M., Kemper B., Kronenberg H. M., Rich A., Potts J. T., Jr Pre-proparathyroid hormone; amino acid sequence, chemical synthesis, and some biological studies of the precursor region. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2616–2620. doi: 10.1073/pnas.75.6.2616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haugen D. A., Armes L. G., Yasunobu K. T., Coon M. J. Amino-terminal sequence of phenobarbital-inducible cytochrome P-450 from rabbit liver microsomes: similarity to hydrophobic amino-terminal segments of preproteins. Biochem Biophys Res Commun. 1977 Aug 8;77(3):967–973. doi: 10.1016/s0006-291x(77)80072-7. [DOI] [PubMed] [Google Scholar]
  16. Haugen D. A., Coon M. J. Induction of multiple forms of mouse liver cytochrome P-450. Evidence for genetically controlled de novo protein synthesis in response to treatment with beta-naphthoflavone or phenobarbital. J Biol Chem. 1976 Mar 25;251(6):1817–1827. [PubMed] [Google Scholar]
  17. Imai Y., Sato R. A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver microsomes of phenobarbital-pretreated rabbits. Biochem Biophys Res Commun. 1974 Sep 9;60(1):8–14. doi: 10.1016/0006-291x(74)90164-8. [DOI] [PubMed] [Google Scholar]
  18. Inouye S., Wang S., Sekizawa J., Halegoua S., Inouye M. Amino acid sequence for the peptide extension on the prolipoprotein of the Escherichia coli outer membrane. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1004–1008. doi: 10.1073/pnas.74.3.1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kreibich G., Debey P., Sabatini D. D. Selective release of content from microsomal vesicles without membrane disassembly. I. Permeability changes induced by low detergent concentrations. J Cell Biol. 1973 Aug;58(2):436–462. doi: 10.1083/jcb.58.2.436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kurtz D. T., Feigelson P. Multihormonal induction of hepatic alpha2u-globulin mRNA as measured by hybridization to complementary DNA. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4791–4795. doi: 10.1073/pnas.74.11.4791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lingappa V. R., Katz F. N., Lodish H. F., Blobel G. A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function. J Biol Chem. 1978 Dec 25;253(24):8667–8670. [PubMed] [Google Scholar]
  22. Matsuura S., Fujii-Kuriyama Y., Tashiro Y. Immunoelectron microscope localization of cytochrome P-450 on microsomes and other membrane structures of rat hepatocytes. J Cell Biol. 1978 Aug;78(2):503–519. doi: 10.1083/jcb.78.2.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Negishi M., Fujii-Kuriyama Y., Tashiro Y., Imai Y. Site of biosynthesis of cytochrome P450 in hepatocytes of phenobarbital treated rats. Biochem Biophys Res Commun. 1976 Aug 23;71(4):1153–1160. doi: 10.1016/0006-291x(76)90774-9. [DOI] [PubMed] [Google Scholar]
  24. Negishi M., Kreibich G. Coordinated polypeptide synthesis and insertion of protoheme in cytochrome P-450 during development of endoplasmic reticulum membranes. J Biol Chem. 1978 Jul 10;253(13):4791–4797. [PubMed] [Google Scholar]
  25. Negishi M., Sawamura T., Morimoto T., Tashiro Y. Localization of nascent NADPH-cytochrome c reductase in rat liver microsomes. Biochim Biophys Acta. 1975 Jan 13;381(1):215–220. doi: 10.1016/0304-4165(75)90203-2. [DOI] [PubMed] [Google Scholar]
  26. Nilsson O. S., DePierre J. W., Dallner G. Investigation of the transverse topology of the microsomal membrane using combinations of proteases and the non-penetrating reagent diazobenzene sulfonate. Biochim Biophys Acta. 1978 Jul 20;511(1):93–104. doi: 10.1016/0005-2736(78)90067-6. [DOI] [PubMed] [Google Scholar]
  27. OMURA T., SATO R. THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. J Biol Chem. 1964 Jul;239:2370–2378. [PubMed] [Google Scholar]
  28. Oda K. I., Joklik W. K. Hybridization and sedimentation studies on "early" and "late" vaccinia messenger RNA. J Mol Biol. 1967 Aug 14;27(3):395–419. doi: 10.1016/0022-2836(67)90047-2. [DOI] [PubMed] [Google Scholar]
  29. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  30. Palmiter R. D., Gagnon J., Ericsson L. H., Walsh K. A. Precursor of egg white lysozyme. Amino acid sequence of an NH2-terminal extension. J Biol Chem. 1977 Sep 25;252(18):6386–6393. [PubMed] [Google Scholar]
  31. Palmiter R. D., Gagnon J., Walsh K. A. Ovalbumin: a secreted protein without a transient hydrophobic leader sequence. Proc Natl Acad Sci U S A. 1978 Jan;75(1):94–98. doi: 10.1073/pnas.75.1.94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Palmiter R. D. Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undergraded polysomes and messenger ribonucleic acid. Biochemistry. 1974 Aug 13;13(17):3606–3615. doi: 10.1021/bi00714a032. [DOI] [PubMed] [Google Scholar]
  33. Palmiter R. D. Prevention of NH2-terminal acetylation of proteins synthesized in cell-free systems. J Biol Chem. 1977 Dec 25;252(24):8781–8783. [PubMed] [Google Scholar]
  34. REMMER H., MERKER H. J. DRUG-INDUCED CHANGES IN THE LIVER ENDOPLASMIC RETICULUM: ASSOCIATION WITH DRUG-METABOLIZING ENZYMES. Science. 1963 Dec 27;142(3600):1657–1658. doi: 10.1126/science.142.3600.1657. [DOI] [PubMed] [Google Scholar]
  35. Ramsey J. C., Steele W. J. A method for visualizing and quantifying the total complement of free and membrane-bound ribosomes in rat liver. Anal Biochem. 1979 Jan 15;92(2):305–313. doi: 10.1016/0003-2697(79)90663-8. [DOI] [PubMed] [Google Scholar]
  36. Ramsey J. C., Steele W. J. A procedure for the quantitative recovery of homogeneous populations of undegraded free and bound polysomes from rat liver. Biochemistry. 1976 Apr 20;15(8):1704–1712. doi: 10.1021/bi00653a018. [DOI] [PubMed] [Google Scholar]
  37. Ramsey J. C., Steele W. J. Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes. Biochem J. 1977 Oct 15;168(1):1–8. doi: 10.1042/bj1680001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Redman C. M., Sabatini D. D. Vectorial discharge of peptides released by puromycin from attached ribosomes. Proc Natl Acad Sci U S A. 1966 Aug;56(2):608–615. doi: 10.1073/pnas.56.2.608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rodriguez Boulan E., Sabatini D. D., Pereyra B. N., Kreibich G. Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins. J Cell Biol. 1978 Sep;78(3):894–909. doi: 10.1083/jcb.78.3.894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rothman J. E., Lodish H. F. Synchronised transmembrane insertion and glycosylation of a nascent membrane protein. Nature. 1977 Oct 27;269(5631):775–780. doi: 10.1038/269775a0. [DOI] [PubMed] [Google Scholar]
  41. Schechter I., Burstein Y., Zemell R., Ziv E., Kantor F., Papermaster D. S. Messenger RNA of opsin from bovine retina: isolation and partial sequence of the in vitro translation product. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2654–2658. doi: 10.1073/pnas.76.6.2654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stäubli W., Hess R., Weibel E. R. Correlated morphometric and biochemical studies on the liver cell. II. Effects of phenobarbital on rat hepatocytes. J Cell Biol. 1969 Jul;42(1):92–112. doi: 10.1083/jcb.42.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Thomas P. E., Korzeniowski D., Ryan D., Levin W. Preparation of monospecific antibodies against two forms of rat liver cytochrome P-450 and quantitation of these antigens in microsomes. Arch Biochem Biophys. 1979 Feb;192(2):524–532. doi: 10.1016/0003-9861(79)90122-x. [DOI] [PubMed] [Google Scholar]
  44. Thomas P. E., Lu A. Y., West S. B., Ryan D., Miwa G. T., Levin W. Accessibility of cytochrome P450 in microsomal membranes: inhibition of metabolism by antibodies to cytochrome P450. Mol Pharmacol. 1977 Sep;13(5):819–831. [PubMed] [Google Scholar]
  45. Toneguzzo F., Ghosh H. P. In vitro synthesis of vesicular stomatitis virus membrane glycoprotein and insertion into membranes. Proc Natl Acad Sci U S A. 1978 Feb;75(2):715–719. doi: 10.1073/pnas.75.2.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Welton A. F., Aust S. D. The effects of 3-methylcholanthrene and phenobarbital induction on the structure of the rat liver endoplasmic reticulum. Biochim Biophys Acta. 1974 Dec 10;373(2):197–210. doi: 10.1016/0005-2736(74)90145-x. [DOI] [PubMed] [Google Scholar]

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